Note: Descriptions are shown in the official language in which they were submitted.
<br/>CA 02682275 2015-10-09<br/>METHODS OF MACROMOLECULAR ANALYSIS<br/>USING NANOCHANNEL ARRAYS<br/>COLOR DRAWINGS<br/>100021 The file of this patent contains at least one drawing/photograph <br/>executed in <br/>color. Copies of this patent with color drawing(s)/photograph(s) will be <br/>provided by the Office <br/>upon request and payment of the necessary fee.<br/>STATEMENT OF GOVERNMENT RIGHTS<br/>100031 This invention was made with U.S. Government support. The Government <br/>may <br/>have certain rights in the invention under National Institutes of Health grant <br/>IR43HG004199-01.<br/>FIELD OF THE INVENTION<br/>[00041 The field of the invention includes nanoscalc devices, and methods of <br/>making <br/>and using such devices, for macromolecular analysis. The field of the <br/>invention also includes <br/>polynucleic acid sizing and structural analysis.<br/>BACKGROUND OF THE INVENTION<br/>[00051 Various scientific and patent publications are referred to herein.<br/>100061 Biomolecules such as DNA or RNA are long molecules composed of <br/>nucleotides, whose linear sequencing is directly related to the genomic and <br/>post-genomic <br/>expression information of the organism.<br/>100071 Biomolecules such as DNA or RNA are long molecules composed of <br/>nucleotides, whose linear sequencing is directly related to the genomic and <br/>post-genomic <br/>expression information of the organism.<br/>100081 In many cases, the mutation or rearrangement of the nucleotide <br/>sequences <br/>during an individual's life span leads to disease states such as genetic <br/>abnormalities or cell <br/>malignancy. In other cases, the small amount of sequence differences among <br/>each individual<br/>- I -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>reflects the diversity of the genetic makeup of the population. Because of <br/>this, different people <br/>have different disease predisposition or respond differently to environmental <br/>stimuli and signals <br/>such as stress or drug treatments. As an example, some patients experience a <br/>positive response <br/>to certain compounds while others experience no effects or even adverse side <br/>effects. Another <br/>area of interest is the response of biomolecules such as DNA to environmental <br/>toxins or other <br/>toxic stimuli such as radiation. Toxic stimuli can lead to programmed cell <br/>death (apoptosis), a <br/>process that removes toxic or non-functioning cells. Apoptosis is <br/>characterized by <br/>morphological changes of cells and nuclei and is often accompanied by the <br/>degradation of <br/>chromosomal DNA.<br/>[0009] Areas of population genomics, comparative/evolution genomics, medical <br/>genomics, environmental or toxicogenomics, and pharmacogenomics studying <br/>genetic diversity <br/>and medical pharmacological implications require extensive sequencing coverage <br/>and large <br/>sample numbers. Knowledge generated from such study would thus be especially <br/>valuable to the <br/>health care and pharmaceutical industry. Cancer genomics and diagnostics in <br/>particular study <br/>genomic instability events leading to tumorigenesis. All these fields would <br/>thus benefit from <br/>technologies enabling fast determination of the linear sequence, structural <br/>pattern changes of <br/>elements/regions of interests on biopolymer molecules such as nucleic acids, <br/>or epigenetic <br/>biomarkers such as methylation patterns along the biopolymers.<br/>[0010] Most genome or epigenome analysis technologies remain too tedious or <br/>expensive for general analysis of large genomic regions or for a large <br/>population. Thus, to <br/>achieve the goal of reducing the genomic analysis cost by at least four orders <br/>of magnitude, the <br/>so-called "$1000 genome" milestone, new paradigm shift technologies enabling <br/>direct molecular <br/>analysis methods are desirable. See "The Quest for the $1,000 Human Genome", <br/>by Nicholas <br/>Wade, The New York Times, July 18, 2006.<br/>[0011] Additionally, it takes on average 7-10 years and 800 million dollars to <br/>bring a <br/>new drug to market. Accordingly, pharmaceutical companies are seeking a safer <br/>and economical <br/>ways to hasten drug development while minimizing the toxicity failure rate.<br/>[0012] Drug compound toxicity can result in damages to DNA in the form of gene <br/>mutations, large scale chromosomal alterations, recombination and numerical <br/>chromosomal <br/>changes. Genotoxicity tests are in vitro and in vivo tests done in bacterial, <br/>mammalian cells and <br/>animals to detect compounds that induce such damages directly or indirectly by <br/>various <br/>mechanisms. The positive compounds could be human carcinogens and/or mutagens <br/>that induce <br/>cancer and/ or heritable defects. A drug can be toxic at different levels, but <br/>drug-induced <br/>mutagenesis of DNA, such as germ line mutations, underlies many long term <br/>adverse effects.<br/>- 2 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>[0013] Despite the guidelines set by government regulatory authorities, there <br/>are cases <br/>of drug toxicity, including the recent issues concerning the COX-2 group of <br/>pain killers. The <br/>toxicity failure rate in the developmental pipeline has remained unimproved <br/>over the years <br/>contributing to the ever increasing costs of the process. During compound <br/>screening, preclinical <br/>testing involves both in vitro and animal assays that assess efficacy and <br/>potential side effects to <br/>predict how the agent will affect humans, but the cost and speed associated <br/>with these <br/>genotoxicity tests have prohibited the wider use and earlier testing to <br/>improve the screening <br/>efficiency. For example, a standard first step for detecting mutagenicity is <br/>the Ames test, <br/>developed almost more than 30 years ago. But even the improved version of the <br/>Ames test takes <br/>requires 2-4 days to process and costs $4,000 to $5,000 per test to complete. <br/>For this reason, <br/>Ames tests are often used in later stages of drug development.<br/>[0014] Among the required genotoxicity test battery, a large component is <br/>evaluation of <br/>chromosomal damage, in vitro or in vivo, involving the tk locus using mouse <br/>lymphoma L5178Y <br/>cells or human lymphoblastoid TK6 cells, the hprt locus using CHO cells, V79 <br/>cells, or L5178Y <br/>cells, or the gpt locus using AS52 cells. The toxicology field uses the <br/>mutation events induced <br/>by compounds at these specific loci as representatives of the entire genome, <br/>hoping the genetic <br/>alterations induced at these loci would be an accurate assessment of the <br/>overall DNA damage of <br/>the genome, for the simplicity of the model system or just sheer lacking of <br/>other efficient and <br/>economic ways of evaluation. In an ideal situation, every time a compound's <br/>exposure time, <br/>dosage, testing cell sampling time or any testing parameter changes, the <br/>entire genome, not just a <br/>few representative gene loci, of the testing cells or system could be <br/>evaluated in detail for <br/>damage information with great efficiency and low cost in a standardized <br/>format. At least, it <br/>would be very beneficial a panel of multi-gene loci, such as one each from <br/>every single <br/>chromosome or key interested regions, could be analyzed without prohibitive <br/>cost and <br/>complexity increase. New technology platform that would allow such <br/>comprehensive pan-<br/>genomic toxicity assessment with efficiency would be greatly desirable.<br/>[0015] In the area of DNA damage assessment, decades-old conventional <br/>cytogenetic <br/>analysis (from karytotyping, G-banding to various forms of FISH) techniques <br/>often rely on a <br/>spread of metaphase DNA, their resolution is limited to the megabase scale. <br/>Interface or fiber-<br/>FISH methods attempt to improve the resolution by using relaxed or partially <br/>stretched DNA but <br/>they are still hard to implement and present challenges when trying to extract <br/>quantitative spatial <br/>information. Powerful as these techniques are, they suffer from poor control <br/>of the processes <br/>since they lack consistency and repeatability, hence are ultimately subject to <br/>the skill of the <br/>technician making them difficult to scale up for faster and cheaper <br/>genotoxicity tests.<br/>- 3 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>[0016] Other recent attempts trying to improve the linearization of individual <br/>DNA <br/>molecules using surface "combing", optical tweezer, fluidic hydrodynamic <br/>focusing flow chip <br/>formats have reflected the desire to further improve the assay consistency, <br/>standardization and <br/>cost feasibility. Unfortunately, the methods of the target DNA elongation are <br/>not inherently well <br/>controlled, the molecule elongation state is often transient, non-uniform and <br/>inconsistent, <br/>deeming complicated analytical process. Such variability limits the <br/>application of this group of <br/>single molecule analysis approach for large scale screening of chromosomal DNA <br/>structural <br/>damages in genotoxicity tests.<br/>[0017] Electrophoresis is also employed to separate polymers of various <br/>molecular <br/>weights based on their varying mobility using gels such as agarose or <br/>polyacrylamide. In the case <br/>of large polymer fragments, the separation time could take hours or even days. <br/>Single cell <br/>electrophoresis assays are routinely used to assess the damage of chromosomal <br/>DNA induced by <br/>toxic agents such as environmental toxins, radiation or agents used in <br/>chemotherapy. In a typical <br/>assay, termed the comet assay, often used in current genotoxicity tests, the <br/>cell is lysed within a <br/>gel matrix and then the DNA is electrophoresed and stained with a fluorescent <br/>dye. During <br/>electrophoresis, DNA fragments migrate away from the cell producing the shape <br/>of a comet tail. <br/>The geometry of the comet tail is related to the number of double stranded and <br/>single stranded <br/>breaks within the chromosomal DNA and thus provides a semi-quantitative <br/>measure of exposure <br/>to toxic agents experienced by the cell. Though this technique offers an <br/>assessment of the degree <br/>of damage, it is difficult to standardize and the data is subject to <br/>interpretation. Also, the <br/>fragments of chromosomal DNA remained entangled and cannot be distinguished <br/>from each <br/>other thus obscuring valuable information regarding the location of breaks or <br/>the size of <br/>individual fragments.<br/>[0018] Other array based approaches such as Comparative Genomic Hybridization <br/>(CGH) have progressed in overcoming some aspects of resolution issues in <br/>detecting unbalanced <br/>genomic structural changes (amplification, deletion not translocation or <br/>inversion) however are <br/>limited to the issues inherit to comparative hybridization. New-generation <br/>sequencing <br/>technologies aim to achieve relatively fast sequence data on individual <br/>genetic molecules in <br/>massive parallel reads; however, molecules analyzed under such techniques must <br/>be fragmented <br/>into relatively short reads (-25 bps) with sequence data generated <br/>computationally, often by <br/>minimizing the tiling path of overlapping reads. A drawback of this approach <br/>is that gross <br/>genetic changes, usually at much larger scale, can often be obscured because <br/>each individual <br/>fragment is removed from the context of the entire genome. This is especially <br/>relevant in the <br/>case of complex genomes with regions of massive repetitive elements and gene <br/>copy number<br/>- 4 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>polymorphism. Accordingly, such techniques lack the ability to provide <br/>information regarding <br/>the whole of a genome, as opposed to a discrete region within the genome.<br/>[0019] Molecular combing techniques have leveraged work in cytogenetics to <br/>generate <br/>more detailed analysis at the single molecule level. In molecular combing, DNA <br/>is elongated by <br/>means of a receding fluid meniscus as a droplet of solution is allowed to dry <br/>on a surface. The <br/>solute will migrate towards the boundaries of the droplet in a phenomenon <br/>known as the 'coffee-<br/>stain' effect (Deegan 1997). In the case of DNA in a buffer solution, the DNA <br/>is tethered to the <br/>surface randomly at the boundaries of a liquid phase and then elongated by the <br/>shear force of the <br/>receding meniscus. Unfortunately, this method of stretching is not inherently <br/>well controlled, <br/>and DNA samples on different microslides can never be "combed" identically, <br/>and there is no <br/>way to predict the degree, uniformity of stretching or placement of the <br/>molecules on a surface. <br/>DNA molecules often overlap each other with imperfect linearization (as they <br/>are not physically <br/>separated), and their ends often recoil upon themselves once they are released <br/>from the meniscus, <br/>leaving incompletely-stretched DNA molecules. Such variability accordingly <br/>limits the <br/>application of the combing approach to large scale screening of patients.<br/>[0020] Other attempts to standardize the linearization of individual DNA <br/>molecules <br/>using fluidic biochip platforms proved relatively inefficient in effecting the <br/>desired linearization. <br/>DNA would often fold back on itself or even retain its free solution Gaussian <br/>coil conformation <br/>(essentially, a ball of yarn). The resolution of such techniques, furthermore, <br/>is poor, because the <br/>elongation of the DNA is often transient, non-uniform and inconsistent, and <br/>images used in <br/>analysis must be captured while the DNA moves at a high enough velocity to <br/>sustain its <br/>elongated state. In addition, because these designs are based around a single <br/>channel through <br/>which flow molecules past an optical detector, their throughput is severely <br/>limited.<br/>[0021] Accordingly, there is a need for efficient determination of the sizes <br/>and <br/>composition of fragments of DNA or other macromolecules by linearizing and <br/>analyzing such <br/>molecules. Such methods would have immediate use in diagnostic and in <br/>treatment applications.<br/>[0022] It would be desirable to use a minimal amount of sample, perhaps as <br/>little as a <br/>single cell. This would greatly advance the ability to monitor the cellular <br/>state and understand <br/>the genesis and progress of diseases such as the malignant stage of a cancer <br/>cell or the degree of <br/>toxicity of a stimulus leading to apoptosis. There is also a related need for <br/>devices capable of <br/>performing such methods.<br/>SUMMARY OF THE INVENTION<br/>- 5 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>[0023] In meeting the challenges for analyzing the size and composition of DNA <br/>segments, the instant invention provides methods for confining, linearizing <br/>and then analyzing <br/>such molecules as well as devices capable of performing such methods.<br/>[0024] First provided are nanofluidic devices capable of manipulating a single <br/>elongated macromolecule, comprising: a substrate comprising a surface and one <br/>or more fluidic <br/>nanochannel segments disposed substantially parallel to the surface, wherein <br/>at least one of the <br/>fluidic nanochannel segments is capable of containing and elongating at least <br/>a portion of a <br/>macromolecule residing within at least a portion of the fluidic nanochannel <br/>segment, and <br/>wherein each of the fluidic nanochannel segments has a characteristic cross-<br/>sectional dimension <br/>of less than about 1000 nm and a length of at least about 10 nm; and at least <br/>one viewing <br/>window, wherein the viewing window is capable of permitting optical inspection <br/>of at least a <br/>portion of the contents of the one or more fluidic nanochannel segments.<br/>[0025] Also provided are methods for characterizing one or more macromolecules <br/>using a nanofluidic device, comprising translocating at least a portion of at <br/>least one region of <br/>the macromolecule through a fluidic nanochannel segment disposed substantially <br/>parallel to a <br/>surface of a substrate, wherein the fluidic nanochannel segment is capable of <br/>containing and <br/>elongating at least a portion of a region of the macromolecule, and wherein <br/>the fluidic <br/>nanochannel segment has a characteristic cross-sectional dimension of less <br/>than about 1000 nm <br/>and a length of at least about 10 nm; and monitoring, through a viewing window <br/>capable of <br/>permitting optical inspection of at least a portion of the contents of the <br/>fluidic nanochannel <br/>segment, one or more signals related to the translocation of one or more <br/>regions of the <br/>macromolecule through the nanochannel; and correlating the monitored signals <br/>to one or more <br/>characteristics of the macromolecule.<br/>[0026] Further provided are devices, comprising A device, comprising: a <br/>substrate <br/>comprising a surface and one or more fluidic nanochannel segments disposed <br/>substantially <br/>parallel to the surface, wherein at least one of the fluidic nanochannel <br/>segments is capable of <br/>containing and elongating at least a portion of a macromolecule residing <br/>within at least a portion <br/>of the fluidic nanochannel segment, and wherein each of the fluidic <br/>nanochannel segments has a <br/>characteristic cross-sectional dimension of less than about 1000 nm and a <br/>length of at least about <br/>nm; and wherein at least a portion of at least one fluidic nanochannel segment <br/>is illuminated <br/>by one or more excitation sources.<br/>[0027] Additionally disclosed are macromolecular analysis devices, comprising <br/>one or <br/>more nanochannels disposed on a surface, one or more of the nanochannels <br/>having a width of <br/>less than about 1000 nm, and one or more of the nanochannels being defined by <br/>one or more<br/>- 6 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>borders and being capable of constraining at least a portion of the <br/>macromolecule so as to <br/>maintain in linear form that portion of the macromolecule.<br/>[0028] Also provided are methods of analyzing macromolecules, comprising <br/>disposing <br/>one or more macromolecules onto a surface having one or more nanochannels <br/>capable of <br/>constraining at least a portion of the macromolecule so as to maintain in <br/>linear form that portion <br/>of the macromolecule; subjecting the one or more macromolecules to a <br/>motivating force so as to <br/>elongate at least a portion of one or more macromolecules within one or more <br/>nanochannels; and <br/>monitoring one or more signals evolved from one or more of the macromolecules.<br/>[0029] The present invention also teaches methods of of fabricating a <br/>macromolecular <br/>analysis device, comprising defining one or more nanochannels on a surface by <br/>disposition of <br/>two or more borders, one or more of the borders being capable of constraining <br/>a macromolecule, <br/>and one or more of the nanochannels having a width of less than about 1000 nm.<br/>BRIEF DESCRIPTION OF THE DRAWINGS<br/>[0030] The summary, as well as the following detailed description, is further <br/>understood when read in conjunction with the appended drawings. For the <br/>purpose of <br/>illustrating the invention, there are shown in the drawings exemplary <br/>embodiments of the <br/>invention; however, the invention is not limited to the specific methods, <br/>compositions, and <br/>devices disclosed. In addition, the drawings are not necessarily drawn to <br/>scale. In the drawings:<br/>[0031] FIG. lA illustrates detection of a macro-molecule flowing through a <br/>nanochannel device where passage of the macromolecule through the nanochannel <br/>is recorded by <br/>exciting features of interest to fluoresce with an excitation source, and then <br/>sensing the <br/>fluorescence with a photon detection device and this fluorescent signal is <br/>then correlated along <br/>the length of the macromolecule;<br/>[0032] FIG. 1B illustrates a cross-sectional view of the device, where light <br/>from an <br/>excitation source illuminates the features of interest as they pass under the <br/>photon detector, <br/>which detector in turn monitors any photons emitted by the illuminated <br/>features;<br/>[0033] FIG. 2A illustrates detection of a macromolecule flowing through a <br/>nanochannel device, whereby the macromolecule is exposed to the excitation <br/>illumination passed <br/>through a slit, where the fluorescent signal is acquired from the region of <br/>the macromolecule in <br/>the nanochannel that is under the slit ¨ by flowing the macromolecule through <br/>the nanochannel, a <br/>stream of fluorescent signals can be collected from the slit that can be used <br/>to determine <br/>characteristic features along the length of the macromolecule, as is shown in <br/>FIG. 2B;<br/>[0034] FIG. 3 illustrates an example of how fluorescent signals from <br/>macromolecules <br/>flowing through nanochannels aquired using a slit can generate a stream of <br/>data;<br/>- 7 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>[0035] FIG. 3A depicts the fluorescent signals of the molecules as they flow <br/>along the <br/>channels, and using a data analysis algorithm, the number of macromolecules <br/>and their lengths <br/>can be determined;<br/>[0036] FIG. 3B illustrates a plot of fluorescent signal intensity versus time <br/>of the <br/>macromolecules in FIG. 3A as they pass by the slit, FIG. 3D illustrates a plot <br/>of fluorescent <br/>signal intensity versus time of the macromolecules in FIG. 3C as they pass by <br/>the slit ¨ in both <br/>both cases, information regarding the distribution of macromolecule size can <br/>be determined from <br/>the detected signal;<br/>[0037] FIG. 4A illustrates an example of a macromolecule flowing through a <br/>nanochannel device, whereby the macromolecule is exposed to excitation <br/>illumination that is <br/>focused on a defined region of the nanochannels ¨ in such an embodiment, the <br/>fluorescent signal <br/>is acquired from the region of the macromolecule in the nanochannel that is <br/>illuminated, and by <br/>flowing the macromolecule through the illuminated region, a stream of <br/>fluorescent signals can be <br/>collected from the macromolecule, FIG. 4B, that can be used to determine <br/>characteristic features <br/>along the length of the macromolecule;<br/>[0038] FIG. 5A illustrates a macromolecule flowing through a nanochannel <br/>device, <br/>whereby the macromolecule is exposed to an excitation illumination source that <br/>is integrated <br/>with the chip device ¨ in this embodiment, the fluorescent signal is acquired <br/>from the region of <br/>the macromolecule in the nanochannel that is illuminated, and by flowing the <br/>macromolecule <br/>through the illuminated region, a stream of fluorescent signals is collected <br/>from the <br/>macromolecule, FIG. 5B, that can be used to determine characteristic features <br/>along the length <br/>of the macromolecule;<br/>[0039] FIG. 6A illustrates an example of a macromolecule flowing into and <br/>being at <br/>least partially elongated by a nanochannel device in which the nanochannels <br/>are covered by a <br/>cap ¨ following elongation, the macromolecule is adhered to the surface and <br/>the cap is removed, <br/>see FIG. 6B, exposing the elongated region of the macromolecule and making the <br/>elongated <br/>region of the macromolecule available for additional analysis, processing, <br/>treatment, and the <br/>like;<br/>[0040] FIG. 7 illustrates a branched nanochannel network in which each <br/>nanochannel <br/>is connected to one or more nanochannels ¨ as the macromolecule flows through <br/>the network, <br/>the macromolecule's degree of elongation is a function of the cross-sectional <br/>dimension of the <br/>nanochannel, and for an example macromolecule flowing through three sucessive <br/>nanochannels <br/>whereby their cross-sectional diameters varies (D3 > D2 > Dl), the <br/>macromolecule's degree of<br/>- 8 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>elongation will also vary (L3 <L2 < L1), similarly the distance between <br/>features of interest on <br/>the macromolecule will vary in a scalable manner (T3 <T2 <T1);<br/>[0041] FIG. 8A is an illustration of labeled macromolucules traversing a <br/>number of <br/>fluidic nanochannel segments, where the segments are disposed in a grid-like <br/>pattern, and where <br/>the DNA molecules are elongated as they traverse the segments -- FIG. 8B <br/>depicts labeled <br/>macromolecules traversing non-linear fluidic nanochannel segments;<br/>[0042] FIG. 9 illustrates DNA molecules elongated in (FIG. 9A) a nanotrench <br/>where <br/>the boundaries of the trench are defined by a topological patterning of the <br/>surface; and (FIG. 9B) <br/>a nanotrack or nanolane where the boundaries of the track are defined by <br/>variations in the surface <br/>properties;<br/>[0043] FIG. 10 illustrates macromolecules elongated in a nanochannel device in <br/>which <br/>the cap material is permeable to agents which can interact with the <br/>macromolecule while the <br/>macromolecule resides within a nanochannel ¨ such a permeable cap can also be <br/>used to pre-<br/>treat nanochannels with agents in order that the agents interact with the <br/>macromolecules once the <br/>macromolecules enter into the pre-treated nanochannels;<br/>[0044] FIG. 11 illustrates various configurations of nanochannel networks, and <br/>depicts <br/>networks where nanochannels are in fluidic communication with each other and <br/>where the <br/>nanochannels are disposed parallel to one another;<br/>[0045] FIG. 12A illustrates DNA fragments of various sizes;<br/>[0046] FIG. 12B is a closer view of the DNA fragments boxed-in in FIG. 12A;<br/>[0047] FIG. 12C depicts the image intensity as a function of position for the <br/>boxed-in <br/>DNA fragments of FIGS. 12A and 12B;<br/>[0048] FIG. 13A depicts several labeled DNA fragments of varying lengths;<br/>[0049] FIG. 13B depicts the image intensity as a function of position for the <br/>DNA <br/>fragments of FIG. 13A;<br/>[0050] FIG. 14 depicts two applications for the disclosed nanochannel devices <br/>and <br/>methods ¨ the left-hand panel of FIG. 14 depicts the use of the nanochannel <br/>device to <br/>characterize a population of macromolecules, which characterization can <br/>include the distribution <br/>of molecule sizes within the population or the concentration of certain <br/>biomarkers within the <br/>group, and the right-hand panel of FIG. 14 depicts the use of the nanochannel <br/>device to <br/>characterize an individual molecule, including the size of the individual <br/>molecule and the spatial <br/>location of biomarkers on the single molecule;<br/>[0051] FIG. 15 is a schematic view of a representative nanochannel device, <br/>wherein <br/>(A) indicates sample inlets, (B) indicates the nanochannels disposed on the <br/>device (C) indicates a<br/>- 9 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>waste region for receiving sample that has flowed through the nanochannels, <br/>and (D) indicates <br/>structures capable of forming electrical or other connections with other <br/>devices, apparatuses, and <br/>the like external to the nanochannel device;<br/>[0052] FIG. 16 is a schematic view of a nanochannel device mating to a plastic <br/>housing <br/>containing one or more connections aligned so as to interface the nanochannel <br/>device with other <br/>devices external to the device -- FIG. 16 also provides a schematic view of an <br/>array of <br/>nanochannels, wherein the nanochannels interface with microfluidics as well as <br/>a set of pillars, <br/>where the pillars are capable of at least partially straightening one or more <br/>macromolecules <br/>before the macromolecules enter the nanochannels;<br/>[0053] FIGS. 17 and 18 are micrographs of patterns formed on a surface having <br/>charged and uncharged regions; the charged regions are marked with indicator <br/>dust; and<br/>[0054] FIG. 19 depicts a nanochannel array wherein macromolecules include <br/>beads <br/>that act to immobilize the macromolecules at the inlet or entry of the <br/>macromolecules ¨ the beads <br/>are sized to obstruct the inlets of the nanochannels.<br/>DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS<br/>[0055] The present invention may be understood more readily by reference to <br/>the <br/>following detailed description taken in connection with the accompanying <br/>figures and examples, <br/>which form a part of this disclosure. It is to be understood that this <br/>invention is not limited to the <br/>specific devices, methods, applications, conditions or parameters described <br/>and/or shown herein, <br/>and that the terminology used herein is for the purpose of describing <br/>particular embodiments by <br/>way of example only and is not intended to be limiting of the claimed <br/>invention. Also, as used in <br/>the specification including the appended claims, the singular forms "a," "an," <br/>and "the" include <br/>the plural, and reference to a particular numerical value includes at least <br/>that particular value, <br/>unless the context clearly dictates otherwise. The term "plurality", as used <br/>herein, means more <br/>than one. When a range of values is expressed, another embodiment includes <br/>from the one <br/>particular value and/or to the other particular value. Similarly, when values <br/>are expressed as <br/>approximations, by use of the antecedent "about," it will be understood that <br/>the particular value <br/>forms another embodiment. All ranges are inclusive and combinable.<br/>[0056] It is to be appreciated that certain features of the invention which <br/>are, for clarity, <br/>described herein in the context of separate embodiments, may also be provided <br/>in combination in <br/>a single embodiment. Conversely, various features of the invention that are, <br/>for brevity, <br/>described in the context of a single embodiment, may also be provided <br/>separately or in any <br/>subcombination. Further, reference to values stated in ranges include each and <br/>every value <br/>within that range.<br/>- 10 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>Terms<br/>[0057] As used herein, the term "channel" means a region defined by borders. <br/>Such <br/>borders may be physical, electrical, chemical, magnetic, and the like. The <br/>term "nanochannel" is <br/>used to clarify that certain channels are considered nanoscale in certain <br/>dimensions.<br/>[0058] As used herein, the term "biomolecule" means DNA, RNA, protein, and <br/>other <br/>molecules of biological origin.<br/>[0059] Nanochannels having diameters below 200 nm have been shown to linearize <br/>couble-stranded DNA molecules, thus preventing the molecule from bending back <br/>on itself and <br/>completely precluding the native Gaussian coil configuration normally assumed <br/>in free solution. <br/>(Cao et at., APL, 2002a) Such conformational constraints are ideal vehicles <br/>for single molecule <br/>DNA analysis. (Cao et at., APL, 2002b). Nanochannels have been shown to <br/>routinely linearize <br/>fragments that are ranged in size from several hundred kilobases to megabases <br/>(Tegenfeldt et at., <br/>PNAS, 2004). Furthermore, the nature of fluidic flow in a nanoscale <br/>environment precludes <br/>turbulence and many of the shear forces that would otherwise fragment long DNA <br/>molecules. <br/>This is especially valuable for macromolecule linear analysis, especially in <br/>molecular analysis of <br/>genomic structural pattern changes with specific probes or non-specific <br/>barcoding labeling <br/>schemes and features of interests such as epigenomic biomarkers of CpG islands <br/>clusters.<br/>[0060] These favorable characteristics further open the possibility of long <br/>range linear <br/>sequencing applications in which intact native DNA is used without <br/>fragmentation or<br/>subcloning. In addition, there is no limit of the read length or capacity as <br/>the parallel or <br/>interwoven nanochannels could be fabricated as long as 30 cm long, with a <br/>density greater than <br/>tens of thousands of channels per cm. Most importantly, entrapping and <br/>linearizing polymers <br/>like genomic DNA in nanochannels that are enclosed or non-enclosed, made by a <br/>well controlled <br/>fabrication or self-assembly approach, would allow ultimately allow the highly <br/>desired <br/>standardization of quantitative measurements of polymers at the single <br/>molecuel level.<br/>[0061] Nanochannels are distinct from nanopores in that nanopores have a very <br/>low <br/>aspect ratio (length/diameter) while nanochannels have a high aspect ratio. <br/>Typically, nanopores <br/>are 0.5 to 100nm in diameter but only a few nm in length. Nanochannels may be <br/>of similar <br/>diameter but are at least lOnm in length.<br/>[0062] A nanochannel's effective diameter is dictated by the radius of <br/>gyration and <br/>persistence length of the polymer to be analyzed so as to ensure complete or <br/>near complete <br/>linearization of the portion of the polymer inside the nanochannel. Semi-<br/>flexible polymer chains<br/>bundle up into a random coil in free solution with a radius of gyration <br/>defined as Rg=(Lp/6)1/2 <br/>wherein L is the calculated contour length and p is the persistence length of <br/>the polymer chain.<br/>- 11 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>A k-DNA segment 16.5 microns in length ¨ and containing approximately 500 <br/>persistence <br/>lengths ¨ has a radius of gyration of approximately 734 nm. Chen, et al., <br/>Probing Single DNA <br/>Molecule Transport Using Fabricated Nanopores, Nano Letters, 2004, 4, 11, 2293-<br/>2298. A 4681 <br/>base-pair double-stranded DNA fragment has a radius of gyration of <br/>approximately 280 nm. <br/>Dietrich, et al., Advances in the Development of a Novel Method to be used in <br/>Proteomics using <br/>Gold Nanobeads, Ultrasensitive and Single-Molecule Detection Technologies, <br/>edited by Jorg <br/>Enderlein, et al, Proc. of SPIE Vol. 6092, 6092C (2006). Thus, a nanochannel <br/>may have a <br/>diameter smaller than twice the radius of gyration of the analyzed polymer <br/>coil. Nanochannels <br/>of such dimension begin to exert entropic confinement on the free fluctuating <br/>polymer coil, <br/>causing it to elongate and/or linearize.<br/>[0063] Biological molecules such as DNA or RNA fragments are long polymers and <br/>their size often carries significant information that is unknown in a <br/>heterogeneous biological <br/>sample. Electrophoresis is usually employed to separate polymers of various <br/>molecular weights <br/>based on their varying mobility using gels such as agarose or polyacrylamide . <br/>In the case of <br/>large polymer fragments, the separation time could take hours or even days. <br/>For the purposes of <br/>this application biomolecules such as DNA, RNA, protein, or other single <br/>molecules are referred <br/>to as macromolecules.<br/>[0064] Long nanochannels with sufficiently small dimensions as described above <br/>are <br/>effective for elongating these polymer chains in a linear form through <br/>entropic confinement, <br/>rendering their apparent contour length quantitatively correlated to their <br/>individual molecular <br/>weight.<br/>[0065] This is especially important for applications such as genotoxicity ¨ a <br/>determination of the genetic damage inflicted by a particular compound or <br/>compounds ¨ in <br/>which the size and sequence of one or more critical chromosomal DNA fragments <br/>carries <br/>important information regarding the stage of apoptosis and level of exposure <br/>to toxic stimuli. <br/>Genotoxicity testing is of particular importance in pharmaceuticals, see <br/>Guidance For Industry <br/>52B Genotoxicity: A Standard Battery for Genotoxicity Testing of <br/>Pharmaceuticals, <br/>International Conference on Harmonisation of Technical Requirements for <br/>Registration of <br/>Pharmaceuticals for Human Use, 1997. It is recommended, see id., that <br/>genotoxicity testing in <br/>pharmaceuticals involve (1) a test for gene mutation in bacteria; (2) an in <br/>vitro test with <br/>cytogenic evaluation of chromosomal damage with mammalian cells or an in vitro <br/>mouse <br/>lymphoma tk assay; and (3) an in vivo test for chromosomal damage using rodent <br/>hematopoetic <br/>cells. Accordingly, a method for efficiently performing genotoxicity testing <br/>would have <br/>immediate applicability to the pharmaceutical industry.<br/>- 12 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>[0066] Determining the size of DNA fragment could provide further information <br/>as to <br/>where factors, caused directly or indirectly by the said stimuli, are <br/>interacting with the long <br/>polymers; or where the damage would occur at specific locations in correlation <br/>to specific <br/>conditions. It has been reported that during apoptosis, chromosomal DNA is <br/>first digested into <br/>fragments that are 50-300 kbps in size. A subsequent digestion step results in <br/>fragments that are <br/><1 kbp (Nagata et at., Cell Death and Diff. 2003).<br/>[0067] In the area of toxicogenomics, single cell electrophoresis assays are <br/>routinely <br/>used to assess the damage of chromosomal DNA induced by toxic agents such as <br/>environmental <br/>toxins, radiation or agents used in chemotherapy. In a typical assay termed <br/>the comet assay, the <br/>cell is lysed within a gel matrix and then the DNA is electrophoresed and <br/>stained with a <br/>fluorescent dye.<br/>[0068] During electrophoresis, DNA fragments migrate away from the cell <br/>producing <br/>the shape of a so-called comet tail. The geometry of the comet tail is related <br/>to the number of <br/>double stranded and single stranded breaks within the chromosomal DNA and thus <br/>provides a <br/>semi-quantitative measure of exposure to toxic agents experienced by the cell. <br/>Though this <br/>technique offers single cell analysis by definition, it is difficult to <br/>standardize and the data is <br/>subject to interpretation. Also, the fragments of chromosomal DNA remain <br/>entangled and <br/>cannot be distinguished from each other, thus obscuring information regarding <br/>the location of <br/>breaks or the size of individual fragments.<br/>[0069] Lastly, DNA damage assessment caused by radiation is another important <br/>field. <br/>Besides cases of accidental exposure to various forms of radiation, more than <br/>half of all cancer <br/>patients receive radiation therapy at some point. Determining the correct <br/>radiation dose to <br/>minimize side effects while retaining maximum effectiveness in killing a tumor <br/>is challenging. <br/>A typical radiation treatment plan is 30 sessions; however, in current <br/>practice a treatment plan is <br/>basically set from the beginning, based on data from the so-called best <br/>treatment for the <br/>"average" patient and not what might be appropriate for each individual. <br/>Finding new <br/>diagnostics and therapeutics to optimize radiation therapy toward personalized <br/>medicine in the <br/>radiation oncology field is a high priority.<br/>[0070] At the molecular level, radiation therapy kills tumor cells by <br/>essentially <br/>breaking up their DNA. Detecting this genetic damage in a manner that could <br/>give physicians <br/>valuable feedback can help adjust subsequent treatment. In current radiation <br/>dosimetry assays, <br/>genomic damage assessment and cell viability after exposure were often assayed <br/>in a relatively <br/>tedious and slow fashion without direct quantitative information of what is <br/>going on inside the <br/>tumor or surrounding healthy cells.<br/>- 13 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>[0071] As applied to radiation therapy, a nanochannel array based device could <br/>physically unwind genomic DNA samples from their natural coiled structure to a <br/>linear form and <br/>analyze the population characteristics such as degree of fragmentation damage. <br/>This method can <br/>monitor changes in the integrity of the DNA samples taken from a tumor and <br/>surrounding tissue <br/>and quantify the damage in an instantaneous fashion to better guide treatment <br/>with "functional" <br/>tumor information.<br/>[0072] In one aspect, the present invention provides nanofluidic devices <br/>capable of <br/>manipulating a single elongated macromolecule, comprising: a substrate <br/>comprising a surface <br/>and one or more fluidic nanochannel segments disposed substantially parallel <br/>to the surface, <br/>wherein at least one of the fluidic nanochannel segments is capable of <br/>containing and elongating <br/>at least a portion of a macromolecule residing within at least a portion of <br/>the fluidic nanochannel <br/>segment, and wherein each of the fluidic nanochannel segments has a <br/>characteristic cross-<br/>sectional dimension of less than about 1000 nm and a length of at least about <br/>10 nm; and at least <br/>one viewing window, wherein the viewing window is capable of permitting <br/>optical inspection of <br/>at least a portion of the contents of the one or more fluidic nanochannel <br/>segments.<br/>[0073] In some embodiments, as shown in FIG. 11, the fluidic nanochannel <br/>segments <br/>that are not fluidically connected to each other, and can in some cases be <br/>disposed essentially <br/>parallel on one another.<br/>[0074] In other embodiments, also as shown in FIG. 11, at least a portion of <br/>the fluidic <br/>nanochannel segments are fluidically connected to each other. In some of these <br/>embodiments, <br/>the fluidic nanochannel segments fluidically connected to each other are <br/>disposed in a branching <br/>pattern or in a grid pattern. Certain patterns of nanochannels can be achieved <br/>by self-assembly <br/>techniques known to those in the art.<br/>[0075] One or more of the fluidic nanochannel segments can, in some cases be <br/>curved <br/>in form, tortuous in form, or even have a varying cross-sectional dimension. <br/>It is contemplated <br/>that not all nanochannels are equivalent in cross-sectional dimension; in some <br/>case, at least one <br/>of the fluidic nanochannel segments comprises a cross-sectional dimension that <br/>is different than <br/>the cross-sectional dimension of another of the fluidic nanochannel segments.<br/>[0076] It is also contemplated, see FIG. 11, that nanochannel segments can be <br/>interconnected or even vary in cross-section.<br/>[0077] Substrates suitable for the present invention include metals, ceramics, <br/>polymers, <br/>glasses, silicons, semiconductors, plastics, dielectrics, SiGe, GaAs, ITO, <br/>fused silica, and the <br/>like. The optimal substrate will be dictated by the needs of the user.<br/>- 14 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>[0078] Suitable fluidic nanochannel segments have a characteristic cross-<br/>sectional <br/>dimension of less than about 500 nm, or of less than about 200 nm, or of less <br/>than about 100 nm, <br/>or even of less than about 50 nm, about 10 nm, about 5 nm, about 2 nm, or even <br/>less than about <br/>than about 0.5 nm.<br/>[0079] A fluidic nanochannel segment suitably has a characteristic cross-<br/>sectional <br/>dimension of less than about twice the radius of gyration of the <br/>macromolecule. In some <br/>embodiments, the nanochannel has a characteristic cross-sectional dimension of <br/>at least about the <br/>persistence length of the macromolecule.<br/>[0080] Fluidic nanochannel segments suitable for the present invention have a <br/>length of <br/>at least about 100 nm, of at least about 500 nm, of at least about 1000 nm, of <br/>at least about 2 <br/>microns, of at least about 5 microns, of at least about 10 microns, of at <br/>least about 1 mm, or even <br/>of at least about 10 mm. Fluidic nanochannel segments are, in some <br/>embodiments, present at a <br/>density of at least 1 fluidic nanochannel segment per cubic centimeter.<br/>[0081] Viewing windows of the invention can comprise a slit, a porthole, a <br/>square, or <br/>any combination thereof. In some configurations, the viewing window is <br/>removable, or <br/>permeable, see FIG. 10. Permeable windows are suitably capable of placing the <br/>contents of one <br/>or more fluidic nanochannel segments into fluid communication with the <br/>environment external to <br/>the fluidic nanochannel segment.<br/>[0082] As shown in FIGS. 9A and 9B, fluidic nanochannel segments may be<br/>characterized as trenches, and some devices comprise a cap capable of covering <br/>at least a portion <br/>of at least one trench. See FIG. 6. In some embodiments, at least a portion of <br/>the cap is <br/>permeable to soluble analytes capable of interaction with a macromolecule <br/>residing in the fluidic <br/>nanochannel segment, FIG. 10, or is removable or even optically transparent. <br/>In some <br/>embodiments, one or more macromolecules are at least partially elongated in <br/>the fluidic <br/>nanochannel segment and remain in a substantially elongated form after the cap <br/>is removed. See <br/>FIG. 6B.<br/>[0083] In other embodiments, FIG. 1, fluidic nanochannel segments are <br/>characterized <br/>as tunnels, and, in some cases can be characterized as a zone bordered by one <br/>or more regions <br/>having a surface chemistry. See FIG. 9B. Suitable surface chemistries includes <br/>a hydrophobic <br/>species, a hydrophilic species, a surfactant, a thiol, an amine, a hydroxyl, <br/>an alcohol, a carbonyl, <br/>a silane, and the like. Other surface chemistries are described elsewhere <br/>herein.<br/>[0084] It is contemplated that one or more fluidic nanochannel segments is in <br/>fluid <br/>communication with one or more fluidic devices, such as conduits, pumps, <br/>filters, screens, <br/>occlusions, gels, heaters, splitters, reservoirs, and the like.<br/>- 15 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>[0085] Macromolecules suitable for use in the device include polymers, double-<br/>stranded DNA, single-stranded DNA, RNA, polypeptides, biological molecules, <br/>proteins, and the <br/>like. Suitable polymers include homopolymers, copolymers, block copolymers, <br/>random <br/>copolymers, branched copolymers, dendrimers, or any combination thereof<br/>[0086] The present devices include, in certain embodiments, one or more <br/>connectors <br/>capable of placing the device in fluid communication with one or more <br/>apparatuses external to <br/>the device; suitable apparatuses include pump, conduits, filters, screens, <br/>gels, heaters, occlusions, <br/>splitters, reservoirs, or any combination thereof.<br/>[0087] Also disclosed are methods for characterizing one or more <br/>macromolecules <br/>using a nanofluidic device, comprising: translocating at least a portion of at <br/>least one region of <br/>the macromolecule through a fluidic nanochannel segment disposed substantially <br/>parallel to a <br/>surface of a substrate, wherein the fluidic nanochannel segment is capable of <br/>containing and <br/>elongating at least a portion of a region of the macromolecule, and wherein <br/>the fluidic <br/>nanochannel segment has a characteristic cross-sectional dimension of less <br/>than about 1000 nm <br/>and a length of at least about 10 nm; and monitoring, through a viewing window <br/>capable of <br/>permitting optical inspection of at least a portion of the contents of the <br/>fluidic nanochannel <br/>segment, one or more signals related to the translocation of one or more <br/>regions of the <br/>macromolecule through the nanochannel; and correlating the monitored signals <br/>to one or more <br/>characteristics of the macromolecule.<br/>[0088] The claimed methods can also include exposing at least one biological <br/>entity to <br/>an agent or agents of interest, to metabolites of such agents, to salts of the <br/>agents, and the like. <br/>Agents include dyes, labels, proteins, enzymes, probes, nucleotides, <br/>oligonucleotides, and similar <br/>species.<br/>[0089] Exposure is accomplished by injecting, treating, spraying, <br/>transfecting, <br/>digesting, immersing, flowing, or applying the agent. As one example, a cell <br/>might could be <br/>incubated in a medium containing a dye agent for a period of time so as to <br/>expose the cell to that <br/>agent.<br/>[0090] Biologial entities suitably subjected to the claimed methods are not <br/>limited to <br/>cells; such entities may also include living creatures, biological molecules, <br/>proteins, and the like. <br/>Components of such entities may also be subjected to the claimed entities.<br/>[0091] In some embodiments, the methods also include isolating one or more <br/>macromolecules from the biological entity. Isolating may be accomplished by <br/>means known to <br/>those of ordinary skill in the art. A non-limiting list of such means <br/>includes, for example,<br/>- 16 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>extracting, lysing, purifying, pulling, manipulating, reacting, distilling, <br/>electrophoresing, and the <br/>like.<br/>[0092] Various macromolecules are suitably subjected to the claimed methods. <br/>Some <br/>of these macromolecules include proteins, single-stranded DNA, double-stranded <br/>DNA, RNA, <br/>siRNA, and the like. Polymers and other chain-structured molecules are also <br/>suitably used in the <br/>claimed methods.<br/>[0093] Macromolecules used in the methods may also be divided the one or more <br/>macromolecules into two or more segments. In some cases, this enables more <br/>efficient <br/>processing or storage of the macromolecules.<br/>[0094] Division of a macromolecule is accomplished by lasing, sonicating, <br/>chemically <br/>treating, physically manipulating, biologically treating, lysing, restricting, <br/>and the like. Those of <br/>ordinary skill in the art will be aware of methods suitable for dividing or <br/>otherwise segmenting <br/>or shortening a given macromolecule<br/>[0095] The methods further include binding a fluorescent label, a radioactive <br/>label, a <br/>magnetic label, or any combination thereof to one or more regions of the <br/>macromolecule. <br/>Binding may be accomplished where the label is specifically complementary to a <br/>macromolecule <br/>or to at least a portion of a macromolecule or other region of interest.<br/>[0096] Translocating includes applying a fluid flow, a magnetic field, an <br/>electric field, <br/>a radioactive field, a mechanical force, an electroosmotic force, an <br/>electrophoretic force, an <br/>electrokinetic force, a temperature gradient, a pressure gradient, a surface <br/>property gradient, a <br/>capillary flow, or any combination thereof. It is contemplated that <br/>translocating includes <br/>controllably moving at least a portion of the macromolecule into at least a <br/>portion of a fluidic <br/>nanochannel segment; moving at least a portion of the macromolecule through at <br/>least a portion <br/>of a fluidic nanochannel segment at a controlled speed and a controlled <br/>direction.<br/>[0097] Monitoring includes displaying, analyzing, plotting, or any combination <br/>thereof <br/>Ways of monitoring signals will be apparent to those of ordinary skill in the <br/>art.<br/>[0098] The one or more monitored signals include optical signals, a radiative <br/>signals, <br/>fluorescent signals, electrical signals, magnetic signals, chemical signals, <br/>or any combination <br/>thereof<br/>[0099] Signals are, in certain embodiments, generated by an electron spin <br/>resonance <br/>molecule, a fluorescent molecule, a chemiluminescent molecule, a radioisotope, <br/>an enzyme <br/>substrate, a biotin molecule, an avidin molecule, an electrical charged <br/>transferring molecule, a <br/>semiconductor nanocrystal, a semiconductor nanoparticle, a colloid gold <br/>nanocrystal, a ligand, a <br/>microbead, a magnetic bead, a paramagnetic particle, a quantum dot, a <br/>chromogenic substrate, an<br/>- 17 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>affinity molecule, a protein, a peptide, a nucleic acid, a carbohydrate, an <br/>antigen, a hapten, an <br/>antibody, an antibody fragment, a lipid, or any combination thereof<br/>[0100] In some embodiments, the molecule is unlabeled and monitored by <br/>infrared <br/>spectroscopy, ultraviolet spectroscopy, or any combination thereof<br/>[0101] The signal is generated by using one or more excitation sources to <br/>induce <br/>fluorescence, chemoluminescence, phosphorescence, bioluminescence, or any <br/>combination <br/>thereof Suitable excitation sources include lasers, visible light sources, <br/>sources of infrared light, <br/>sources of ultraviolet light, or any combination thereof<br/>[0102] Correlating comprises determining the features of a distinct <br/>macromolecule or a <br/>portion thereof from a population of macromolecules by partial or full <br/>elongation of the <br/>macromolecule in a fluidic nanochannel segment. In some embodiments, at least <br/>a portion of the <br/>macromolecule is stationary during the monitoring.<br/>[0103] It is contemplated that in some cases, at least a portion of the <br/>macromolecule is <br/>translocated within at least a portion of the fluidic nanochannel segment more <br/>than one time. <br/>Such translocation allows for multiple analyses of the same region of a given <br/>macromolecule.<br/>[0104] Correlating suitably includes determining the length of at least a <br/>portion of the <br/>macromolecule, determining the apparent partially elongated length of at least <br/>a portion of the <br/>macromolecule as confined within one or more fluidic nanochannel segments. The <br/>apparent <br/>partially elongated length is determined as the linear distance along the <br/>fluidic nanochannel <br/>segment within which a partially elongated macromolecule is confined.<br/>[0105] It is contemplated that correlating also includes determining the <br/>identity of one <br/>or more components of the macromolecule or determining the sequence of one or <br/>more <br/>components of the macromolecule, or determining the presence of one or more <br/>modifications to <br/>at least a portion of the macromolecule, or any combination thereof .<br/>[0106] Correlating is performed by automated means, computerized means, <br/>mechanical <br/>means, manual means, or any combination thereof Correlating includes one or <br/>more algorithms <br/>for characterizing a duplex nucleic acid molecule based on observed signal <br/>modulations through <br/>the detection region of a nanochannel, wherein said algorithm is present on a <br/>computer readable <br/>medium.<br/>[0107] It is contemplated that he one or more characteristics of the <br/>macromolecule are <br/>one or more target features present on at least a portion of the <br/>macromolecule. Suitable target <br/>features include epigenetic factors, such as methylation patterns.<br/>[0108] Target features also include one or more genomic structures, including <br/>the <br/>position of one or more particular molecular sequences, SNPs, haplotypes, <br/>repetitive elements,<br/>- 18 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>copy numbers polymorphisms, a change in one or more loci on a DNA molecule, <br/>open reading <br/>frames, introns, exons, regulatory elements, or any combination thereof Target <br/>features also <br/>include compound/drug binding sites/complex, DNA repairing or cleaving binding <br/>sites/complex, SiRNA or anti-sense nucleotides binding sites/complex, <br/>transcription/regulatory <br/>factor binding sites/complex, restriction enzyme binding/cleaving <br/>sites/complex, or any other <br/>genetically engineered or chemically modified binding sites/complexes, or any <br/>combination <br/>thereof<br/>[0109] The present methods can, in some embodiments, further include <br/>contacting a <br/>macromolecule with a first labeled probe of known length Li, wherein the first <br/>labeled probe is <br/>complementary to a control genomic sequence whose copy number is known, and <br/>with a second <br/>labeled probe of known length L2, wherein the second labeled probe is specific <br/>to a nucleotide <br/>sequence of interest; introducing the macromolecule into at least a portion of <br/>the fluidic <br/>nanochannel segment; elongating the labeled macromolecule within the fluidic <br/>nanochannel <br/>segment;detecting binding between the first labeled probe and the genomic <br/>control sequence and <br/>between the second labeled probe and the nucleotide sequence of interest; and <br/>ascertaining the <br/>total length of the hybridization signals that correspond to the first labeled <br/>probe (Si) and the <br/>second labeled probe (S2).<br/>[0110] The present methods further include calculating the copy number of the <br/>nucleotide sequence of interest. The the copy number is calculated by <br/>calculating the ratios Ni = <br/>Si/Li and N2 = 52/L2, wherein Ni corresponds to the copy number of the genomic <br/>control <br/>sequence and N2 corresponds to the copy number of the nucleotide sequence of <br/>interest. It is <br/>contemplated that the copy number of the control sequence is an integer, and <br/>that the difference <br/>between N2 and Ni indicates an abnormality in the genome being analyzed.<br/>[0111] The methods further contemplate that the control genomic sequence <br/>includes <br/>separate portions whose total length per genome is known, wherein the sequence <br/>of interest <br/>comprises separate portions whose length per normal gene is known, and wherein <br/>a significant <br/>difference between N2 and Ni indicates a genetic abnormality in the genome.<br/>[0112] In some embodiments, the nucleotide sequence of interest can relate to <br/>a <br/>trisomy-linked chromosome, wherein the control genomic sequence is from a <br/>chromosome other <br/>than the trisomy-linked chromosome, and wherein a N2/N1 ratio of approximately <br/>1.5 indicates <br/>a trisomic genotype. In other embodiments, the nucleotide sequence of interest <br/>comprises a <br/>deletion of a portion of a genome. In still other embodiments, the nucleotide <br/>sequence of interest <br/>comprises a repeating sequence.<br/>- 19 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>[0113] In some aspects, the present method includes embodiments wherein the <br/>control <br/>genomic sequence and the nucleotide sequence of interest are identical for a <br/>given genome, and <br/>wherein one or more different genomes are analyzed within one or more fluidic <br/>nanochannel <br/>segments so as to determine the respective quantities of each genome present.<br/>[0114] It is contemplated that the N2/N1 ratio has a statistical error of less <br/>than 20%.<br/>[0115] It is further contemplated that the methods include embodiments where <br/>the <br/>control genomic sequence and nucleotide sequence of interest are from the same <br/>genome, or <br/>even where the control genomic sequence is from the same chromosome as the <br/>nucleotide <br/>sequence of interest.<br/>[0116] The instant methods can further include so-called flanked probes, <br/>labeling <br/>regions of a sample nucleotide at either end of a nucleotide zone of interest <br/>and regions of a <br/>control nucleotide at either end of a nucleotide zone of interest. In such <br/>embodiments, the <br/>methods include (a) introducing the labeled nucleotides into separate fluidic <br/>nanochannel <br/>segments having a cross-sectional diameters sufficient to at least <br/>substantially elongate the <br/>labeled nucleotides, (b) determining the distance between the labels on the <br/>sample nucleotide and <br/>the control nucleotide, respectively, and repeating steps (a) and (b) one or <br/>more times so as to <br/>further linearize the sample and control nucleotides and so as to obtain <br/>additional information <br/>regarding the distance between the labels on the control and sample <br/>nucleotides as the <br/>nucleotides elongate.<br/>[0117] These embodiments further include determining the length of the zone of <br/>interest on the sample nucleotide by comparing the distance between the labels <br/>on the control <br/>and sample nucleotides, wherein a difference between the distance between the <br/>labels on the <br/>control and sample nucleotides indicates an abnormality in the zone of <br/>interest on the sample <br/>nucleotide.<br/>[0118] Further provided are devices, comprising: a substrate comprising a <br/>surface and <br/>one or more fluidic nanochannel segments disposed substantially parallel to <br/>the surface, wherein <br/>at least one of the fluidic nanochannel segments is capable of containing and <br/>elongating at least a <br/>portion of a macromolecule residing within at least a portion of the fluidic <br/>nanochannel segment, <br/>and wherein each of the fluidic nanochannel segments has a characteristic <br/>cross-sectional <br/>dimension of less than about 1000 nm and a length of at least about 10 nm; and <br/>wherein at least a <br/>portion of at least one fluidic nanochannel segment is illuminated by one or <br/>more excitation <br/>sources.<br/>[0119] Suitable fluidic nanochannel segments and patterns and dimensions <br/>thereof are <br/>described elsewhere herein. Suitable substrates are also described elsewhere <br/>herein.<br/>- 20 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>[0120] It is contemplated that the present devices include, in some <br/>embodiments, a <br/>viewing window disposed between the the illumninated fluidic nanochannel <br/>segment and the <br/>illumination source, wherein the viewing window comprises a slit, and, in some <br/>embodiments, is <br/>removable. It is also contemplated that the viewing window is capable of <br/>placing the contents of <br/>one or more fluidic nanochannel segments into fluid communication with the <br/>environment <br/>external to the fluidic nanochannel segment.<br/>[0121] Nanochannel segments are characterized as trenches, which trenches are <br/>described elsewhere herein. Caps suitable for covering such trenches are also <br/>described <br/>elsewhere herein, and it is contemplated that one or more macromolecules are <br/>at least partially <br/>elongated in the fluidic nanochannel segment, and remain in a substantially <br/>elongated form after <br/>the cap is removed.<br/>[0122] Fluidic nanochannels are also characterized as a zone bordered by one <br/>or more <br/>regions having a surface chemistry, which fluidic nanochannels are described <br/>elsewhere herein.<br/>[0123] One or more fluidic nanochannel segments is in fluid communication with <br/>one <br/>or more suitable fluidic devices, which are described elsewhere herein, and <br/>include a screen, an <br/>occlusion, a gel, a heater, a splitter, a reservoir, or any combination <br/>thereof.<br/>[0124] In some embodiments, the devices include one or more obstacles situated <br/>in <br/>proximity to one or more nanochannels. Such obstacles may assist in unfolding <br/>or unraveling <br/>macromolecules to enhance the ability of a macromolecule to enter into the <br/>nanochannel.<br/>[0125] Macromolecules suitable for use in the present invention are described <br/>elsewhere herein. As described elsewhere, the devices may include comprising <br/>one or more <br/>connectors capable of placing the device in fluid communication with one or <br/>more apparatuses <br/>external to the device. Suitable apparatuses are described elsewhere herein.<br/>[0126] Excitation sources suitable for use in the device include lasers, <br/>halogen lights, <br/>mercury lamps, sources of infrared light, source of ultraviolet light, diodes, <br/>waveguides, <br/>radioactive sources, or any combination thereof Devices can further include <br/>one or more filters <br/>capable of transmitting a spectrum of excitation source light.<br/>[0127] The portion of the at least one illuminated fluidic nanochannel segment <br/>illuminated by one or more excitation sources is characterized as being one or <br/>more slits, as one <br/>or more circular spots, ovals, polygons, or any combination thereof.<br/>[0128] Suitable excitation sources are capable of being scanned across at <br/>least a portion <br/>of at least one fluidic nanochannel segment. In some embodiments, the device <br/>includes one or <br/>more excitation sources.<br/>-21 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>[0129] Devices suitably include a detector disposed so as to be capable of <br/>receiving an <br/>optical signal originating from within one or more illuminated fluidic <br/>nanochannel segments.<br/>[0130] Suitable detectors include a charge coupled device (CCD) detection <br/>system, a <br/>complementary metal-oxide semiconductor (CMOS) detection system, a photodiode <br/>detection <br/>system, a photo-multiplying tube detection system, a scintillation detection <br/>system, a photon <br/>counting detection system, an electron spin resonance detection system, a <br/>fluorescent detection <br/>system, a photon detection system, an electrical detection system, a <br/>photographic film detection <br/>system, a chemiluminescent detection system, an enzyme detection system, an <br/>atomic force <br/>microscopy (AFM) detection system, a scanning tunneling microscopy (STM) <br/>detection system, <br/>a scanning electron microscopy (SEM) detection system, an optical detection <br/>system, a nuclear <br/>magnetic resonance (NMR) detection system, a near field detection system, a <br/>total internal <br/>reflection (TIR) detection system, a patch clamp detection system, a <br/>capacitive detection system, <br/>or any combination thereof<br/>[0131] Also disclosed are macromolecular analysis devices. The disclosed <br/>devices <br/>includeone or more nanochannels disposed on a surface, with one or more of the <br/>nanochannels <br/>having a width of less than about 1000 nm, and one or more of the nanochannels <br/>being defined <br/>by one or more borders and being capable of constraining at least a portion of <br/>the macromolecule <br/>so as to maintain in linear form that portion of the macromolecule.<br/>[0132] Nanochannels suitably have a length in the range of from about 10 nm to <br/>about <br/>cm, or from about 100 nm to about 1 cm. While nanochannels may be straight, <br/>parallel, <br/>interconnected, curved, or bent, nanochannels of the instant invention <br/>suitably include at least <br/>one essentially straight portion in the length of from about 10 nm to about <br/>100 cm, or in the <br/>range of from about 100 nm to about 10 cm, or even from about 1 mm to about 1 <br/>cm. As an <br/>example, the claimed invention includes embodiments wherein nanochannels <br/>arranged in a back-<br/>and-forth, radiator-type pattern on a surface.<br/>[0133] The width of nanochannels is suitably less than 1000 nm, or less than <br/>500 nm, <br/>or less than 50 nm. In some embodiments, the nanochannels suitably have a <br/>width of less than <br/>about 10 nm, or even less than about 5 nm.<br/>[0134] As discussed, two or more nanochannels according to the present <br/>invention may <br/>be interconnected. A nanochannel may have a constant cross-section or may vary <br/>in cross-<br/>section, depending on the user's needs.<br/>[0135] Borders that define the nanochannels of the present invention have <br/>various <br/>configurations. A border may suitably be a physical wall, a ridge, or the <br/>like. Alternatively, a <br/>border includes an electrically charged region, a chemically-treated region, a <br/>region of magnetic<br/>- 22 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>field, and the like. Hydrophobic and hydrophilic regions are considered <br/>especially suitable <br/>borders. In some cases, borders are formed from differing materials -- e.g., <br/>strips of glass, <br/>plastic, polymer, or metal. In other embodiments, borders are formed by self-<br/>assembling <br/>monolayers (SAMs). In other embodiments, the nanochannels are of an inverse <br/>construction <br/>wherein exposed surface defines the borders of the nanochannel, and the <br/>central lane of the <br/>channel is qualitatively different from the exposed bordering surface. <br/>Nanochannels are suitably <br/>capable of confining at least a portion of a macromolecule so as to elongate <br/>or unfold that <br/>portion of the macromolecule. For example, a macromolecule that is hydrophilic <br/>may be <br/>elongated by placement or disposition within a nanochannel bounded by <br/>hydrophobic borders. <br/>In this example, the macromolecule will be constrained by the borders and will <br/>become <br/>elongated.<br/>[0136] Surfaces suitable for the disclosed devices include glass, ceramics, <br/>silicon, <br/>metals, polymers, and the like. Surfaces will be chosen according to the <br/>user's needs, and as will <br/>be apparent to those of ordinary skill in the art, certain surfaces will be <br/>optimally amendable to <br/>various chemical or other treatments needed to define border regions on such <br/>surfaces.<br/>[0137] The claimed devices also include a viewing window disposed above at <br/>least a <br/>portion of at least one nanochannel. Such viewing windows may be permeable to <br/>one or more <br/>macromolecules. As an example, a viewing window may include one or more pores, <br/>holes, <br/>channels, or nanochannels, any of which will enable macromolecules to move in <br/>three <br/>dimensions in the claimed devices. Such three-dimensional configurations <br/>permit introduction <br/>and routing of macromolecules in a number of directions and, in some <br/>embodiments, enable <br/>simultaneous viewing of multiple regions of macromolecules within the claimed <br/>devices.<br/>[0138] The disclosed inventions also include detectors. Such detectors are <br/>suitably able <br/>to monitor or capture a signal evolved from a molecule within the claimed <br/>devices; which <br/>detectors include CCD cameras or photon-counter devices.<br/>[0139] The claimed inventions also provide methods of analyzing <br/>macromolecules. <br/>The methods include disposing one or more macromolecules onto a surface having <br/>one or more <br/>nanochannels capable of constraining at least a portion of the macromolecule <br/>so as to maintain in <br/>linear form that portion of the macromolecule, subjecting the one or more <br/>macromolecules to a <br/>motivating force so as to elongate at least a portion of one or more <br/>macromolecules within one or <br/>more nanochannels, and monitoring one or more signals evolved from one or more <br/>of the <br/>macromolecules.<br/>[0140] Macromolecules are suitably disposed onto a surface by comprises <br/>dispensing, <br/>dropping, flowing, and the like. Macromolecules are suitably carried in a <br/>fluid, such as water, a<br/>- 23 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>buffer, and the like, to aid their disposition onto the surfaces. The carrier <br/>fluid is chosen <br/>according to the needs of the user, and suitable carrier fluids will be known <br/>to those of ordinary <br/>skill in the art.<br/>[0141] In some embodiments, one or more macromolecules are disposed at least <br/>partially within one or more nanochannels.<br/>[0142] Suitable motivating forces include pressure gradients, magnetic fields, <br/>electric <br/>fields, receding menisci, surface tension forces, thermal gradients, pulling <br/>forces, pushing forces, <br/>and the like. Other manners of applying a force to macromolecules will be <br/>known to those of <br/>ordinary skill in the art, which manners include optical traps, optical <br/>tweezers, physical probes, <br/>atomic force microscopes, and the like. Motivating forces may be constant, <br/>variable, alternating, <br/>and the frequency and intensity of a motivating force will depend on the <br/>user's needs.<br/>[0143] In some embodiments, one or more macromolecules is tethered to the <br/>surface <br/>for analysis. Tethering may be accomplished by biotin-avidin bonds, by <br/>interactions between <br/>gold and thio- groups, and by antibody-antigen or antibody-epitope <br/>interactions. Users of <br/>ordinary skill in the art will be aware of suitable ways to tether molecules <br/>to surfaces.<br/>[0144] In other embodiments, a macromolecule is at least partially immobilized <br/>by a <br/>dynamic force. For example, a macromolecule may include a bead at one end, <br/>which bead is <br/>larger in diameter than the cross-section of a particular nanochannel. <br/>Application of fluid flow to <br/>such a macromolecule will result in the macromolecule's bead being stuck at <br/>one end of the <br/>nanochannel so as to immobilize the macromolecule extending into at least a <br/>portion of the <br/>nanochannel. In such embodiments, the macromolecule may be released from the <br/>nanochannel <br/>by application of an opposing motivating force, e.g., by reversing the <br/>direction of the fluid flow <br/>field. Magnetic and electric fields are also suitably used to immobilize <br/>macromolecules in <br/>nanochannels, which field are easily reversed to free such immobilized <br/>macromolecules. In such <br/>a way, a given set of nanochannels may be re-used to analyze a given <br/>macromolecule multiple <br/>times or be recycles to analyze a different macromolecule or sets of <br/>macromolecules.<br/>[0145] Monitoring a signal evolved from a macromolecule is accomplished by, <br/>inter <br/>alia, recording, plotting, or displaying the signal; monitored signals are <br/>suitably derived from a <br/>portion of a macromolecule that is in substantially linear form within a <br/>nanochannel. The <br/>monitoring may be performed through a viewing window or by directly <br/>interrogating one or <br/>more macromolecules.<br/>[0146] The disclosed methods also include analyzing one or more evolved <br/>signals, <br/>which analysis suitably includes correlating one or more monitored signals to <br/>one or more <br/>characteristics of one or more macromolecules. Correlating could include, for <br/>example, relating<br/>- 24 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>the existence of a particular signal to the existence of a particular mutation <br/>on a segment of <br/>DNA.<br/>[0147] Also provided are methods of fabricating a macromolecular analysis <br/>devices. <br/>These methods include defining one or more nanochannels on a surface by <br/>disposition of two or <br/>more borders, where one or more of the borders being capable of constraining a <br/>macromolecule, <br/>and one or more of the nanochannels has a width of less than about 1000 nm.<br/>[0148] Nanochannels formed by the instant methods may have widths of less than <br/>500 <br/>nm, less than 100 nm, less than 50 nm, or even less than 10 nm. The optimal <br/>width of a <br/>nanochannel will be dictated by the needs of the user and by the <br/>macromolecules under study.<br/>[0149] Disposition of borders is accomplished by, inter alia, rendering <br/>electrically <br/>charged at least a portion of the surface, rendering at least a portion of the <br/>surface hydrophobic, <br/>rendering at least a portion of the surface hydrophilic, rendering at least a <br/>portion of the surface <br/>magnetic, or any combination thereof. In one embodiment, disposition of is <br/>accomplished by <br/>contacting at least a portion of the surface with a mold having a surface <br/>profile that comprises a <br/>surface profile that is complementary to the desired pattern of borders or <br/>nanochannels. Molds <br/>suitable for the present invention comprise one or more nanoscale features, <br/>and may be <br/>fabricated by methods known to those skilled in the art.<br/>[0150] One exemplary embodiment is shown in FIG. 9B, which figure illustrates <br/>nanochannels or nanolanes defined by borders of Surface B ¨ which may be a <br/>hydrophobic <br/>surface ¨ and lanes of Surface A, which surface may be hydrophilic or other <br/>surface different <br/>from Surface B. Similar borders may also be used to define more intricate <br/>patters of <br/>nanochannels, such as those shown in FIG. 7.<br/>[0151] For example, a mold or other substrate comprising nanochannels can be<br/>contacted with a hydrophobic compound. The mold is then contacted with a <br/>hydrophilic surface, <br/>leaving behind hydrophobic patches on the surface that act as borders, <br/>defining nanochannels on <br/>the surface that correspond to the nanochannel pattern on the mold. Molds or <br/>other patterns may <br/>also be used to effect regions of electric charge or of magnetic fields. This <br/>is accomplished by, <br/>inter alia, contacting the mold with a charge-carrying species, a hydrophobic <br/>species, a <br/>hydrophilic species, a magnetic species, a ferromagnetic species, or any <br/>combination thereof. <br/>Exemplary patterns are shown in FIGS. 17 and 18, which patterns were produced <br/>by disposing <br/>regions of charge on substrates and highlighting those regions of charge by <br/>spreading an <br/>indicator dust over the substrates that bound to the charged regions and <br/>removing the unbound <br/>dust.<br/>EXAMPLES AND OTHER ILLUSTRATIVE EMBODIMENTS<br/>- 25 -<br/><br/>CA 02682275 2014-07-07<br/>[01521 General Procedures. Deposition of capping material was provided by <br/>sputtering, CVD, e¨beam evaporation with a tilted sample wafer at various <br/>angles. This step was <br/>used to both reduce the nanochannel diameter and provide a cap.<br/>[01531 In most cases, 100-340 nm of Si02 was deposited onto the channel <br/>openings.<br/>Effective sealing was achieved with various deposition conditions that were <br/>tested. At gas <br/>pressure of 30 mTorr, RF power of ¨900 W, and DC bias of 1400 V, a deposition <br/>rate of-.9 <br/>nm/min was achieved, At lower pressure of 5 mTorr, the deposition rate was <br/>increased to an <br/>estimated 17 nm/min. Filling material was deposited on the nanochannel opening <br/>by sputtering at <br/>mTorr. Further details about making nanochannel arrays and devices can be <br/>found in U.S. <br/>Patent Application Pub. Nos. US 2004-0033515 Al and US 2004-0197843 Al.<br/>101541 Example 1: A silicon substrate having a plurality of parallel linear <br/>channels <br/>that had an 100 nm trench width and a 100 nm trench height was provided. These <br/>channel <br/>openings were sputtered at a gas pressure of 5 mTorr according to the general <br/>procedures given <br/>above. The sputter deposition time was 10-25 minutes to provide a nanochannel <br/>array that can <br/>range from not completely sealed to completely sealed. Silicon dioxide was <br/>deposited by an e-<br/>beam (thermo) evaporator (Temcscal BJD-1800) onto the substrate. The substrate <br/>was placed at <br/>various angles incident to the depositing beam from the silicon dioxide source <br/>target; the <br/>deposition rate can be set to about 3 nm/minute and 150 nm of sealing material <br/>was deposited in <br/>about 50 minutes. The angle of the incident depositing beam of sealing <br/>material could be varied <br/>to reduce the channel width and height to less than 150 nm and 150 nm, <br/>respectively, and to <br/>substantially sealed by providing shallow tangential deposition angles.<br/>101551 Example 2: In this example, a nanochannel array was contacted with a <br/>surface-<br/>modifying agent. A nanochannel array made according to Example 1 can be <br/>submerged in a <br/>surface-modifying agents solutions containing polyethelyene glycol inside a <br/>vaccum chamber to <br/>facilitate wetting and treatment of the channels and degas the air bubbles <br/>that might be trapped <br/>inside the nanochannels.<br/>[01561 Example 3: This example describes how to provide a sample reservoir <br/>with a <br/>nanochannel array to provide a nanofluidic chip. A nanochannel array having <br/>100 nm wide, 100 <br/>run deep nanochannels was made according to general procedures of Example 1. <br/>The <br/>nanochannel array was spin-coated with a photoresist and imaged with a <br/>photomask to provide <br/>regions on opposite ends of the channel array. The exposed areas were etched <br/>using reactive ion <br/>etching to expose the nanochannel ends and to provide a micron-deep reservoir <br/>about a <br/>millimeter wide on the opposite ends of the channels at the edge of the <br/>substrate.<br/>- 26 -<br/><br/>CA 02682275 2009-09-25<br/>WO 2008/121828 PCT/US2008/058671<br/>[0157] Example 4: This example describes how to fill a nanofluidic chip with a <br/>fluid <br/>containing DNA macromolecules to analyze the DNA. A cylindrical-shaped plastic <br/>sample-<br/>delivery tube of 2 mm diameter was placed in fluid communication with one of <br/>the reservoirs of <br/>the nanochannel array of Example 3. The delivery tube was connected to an <br/>external sample <br/>delivery/collection device, which can be in turn connected to a pressure <br/>/vaccum generating <br/>apparatus. The nanochannels were wetted using capillary action with a buffer <br/>solution. A buffer <br/>solution containing stained for example lambda phage macromolecules (lambda <br/>DNA) were <br/>introduced into the nanochannel array by electric field (at 1-50 V/cm); the <br/>solution concentration <br/>was 0.05-5 microgram/mL and the lambda DNA was stained at a ratio of 10:1 base <br/>pair/dye <br/>with the dye TOTO-1 (Molecular Probes, Eugene, Oregon). This solution of <br/>stained DNA was <br/>diluted to 0.01-0.05microgram/mL into 0.5xTBE (tris-boroacetate buffer at pH <br/>7.0) containing <br/>0.1M of an anti-oxidant and 0.1% of a linear polyacrylamide used as an anti-<br/>sticking agent.<br/>[0158] Example 5: This example describes how to image DNA whole or substantial <br/>parts of macromolecules linearized within nanochannels. The DNA macromolecules <br/>were <br/>fluorescently labeled and flowed into the nanochannels according to the <br/>procedures discussed in <br/>Example 4. An excitation light source such as a 100W halogen lamp was focused <br/>through a 60X <br/>lens onto the nanochannels thereby exciting DNA molecules within the field of <br/>view. <br/>Fluorescent light emission from the TOTO-1 dye molecules is collected through <br/>the lens, was <br/>reflected by a dichroic filter and passed through a filter that allows <br/>transmission of the <br/>wavelength band emitted by TOTO-1. The light was detected using a CCD camera <br/>thus <br/>producing an image of the DNA molecules in the field of view.<br/>[0159] Example 6: This example describes how to detect DNA macromolecules as <br/>they pass through a detection area that is smaller than the end-to-end <br/>physical length of DNA <br/>molecules linearized within nanochannels. DNA was stained and flowed into the <br/>nanochannels <br/>as described in Example 4. The detection area was constrained by defining a <br/>narrow slit through <br/>which excitation light can pass. The slit was defined using a 100 nm film of <br/>aluminum deposited <br/>on top of the nanochannels and then opening a 1 micron slit in the aluminum <br/>using <br/>photolithography and chlorine plasma etching. As the DNA passed through the <br/>part of the <br/>nanochannel under the aluminum slit, it was exposed to the excitation light <br/>and emits fluorescent <br/>light. The fluorescent emission was collected as described in Example 5 but <br/>detected using a <br/>photomultiplier tube (PMT). The PMT registered a signal until the DNA molecule <br/>completely <br/>passed by the slit. By correlating the speed at which DNA moves past the slit <br/>(typically 1-100 <br/>microns/sec) to the length of time that a signal is detected, the size of the <br/>DNA molecule is <br/>determined.<br/>-27 -<br/>
